P27 prevents cellular migration

ABSTRACT

This invention provides methods of preventing cellular migration and of treating cardiovascular diseases and tumor metastasis by increasing cyclin-dependent kinase inhibitor p27 activity, and methods of identifying chemical compounds for use in such treatments.

[0001] The invention disclosed herein was made with Government supportunder grant numbers RO1HL56180, RO1A139794, and RO3TW00949 from theNational Institutes of Health, U.S. Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referencedin parentheses by author and year. Full citations for these referencesmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

[0003] Vascular smooth muscle cell (SMC) migration is believed to play amajor role in the pathogenesis of many vascular diseases, such asatherosclerosis and restenosis after both percutaneous transluminalangioplasty (PTCA) and coronary stenting (Schwartz, 1997). In normalblood vessels, the majority of SMC reside in the media or middle coat ofthe vessel, where they are quiescent and possess a “contractile”phenotype, characterized by the abundance of actin- andmyosin-containing filaments. In disease states, SMCs migrate from themedia to the intima or inner coat of the blood vessel. The process ofSMC migration in pathological states involves the synthesis ofextracellular matrix, protease enzymes, growth factors such asplatelet-derived growth factor (PDGF) and basic fibroblast growth factor(bFGF), and cytokines that further contribute to proliferation andmigration (Clowes and Schwartz, 1985; Ferns et al., 1991; Grotendorst etal., 1981; Ihnatowycz et al., 1981; Jawien et al., 1992). Fibroblastgrowth factor-2 (FGF-2) appears to modulate SMC migration by changingextracellular matrix (ECM)-β1 integrin interactions (Pickering et al.,1997). FGF-2 augments SMC surface expression of α2β1, α3β1 and αvβ1integrins, thereby resulting in enhanced cellular motility throughdisassembly of the α-actin stress fiber network (Pickering et al.,1997).

[0004] Rapamycin, a macrolide antibiotic, inhibits SMC proliferationboth in vitro and in vivo by blocking cell cycle progression at thetransition between the first gap (G1) and DNA synthesis (S) phases (Caoet al., 1995; Gallo et al., 1999; Gregory et al., 1993; Marx et al.,1995). The inhibition of cellular proliferation is associated with amarked reduction in cell cycle dependent kinase activity and inretinoblastoma protein phosphorylation in vitro (Marx et al., 1995) andin vivo (Gallo et al., 1999). Down-regulation of the cyclin-dependentkinase inhibitor (CDKI) p27^(kip1) by mitogens is blocked by rapamycin(Kato et al., 1994; Nourse et al., 1994). Pretreatment of rat and humanSMC with rapamycin (2 nM) for 48 hours inhibited PDGF-induced SMCmigration in a modified Boyden chamber. However, acute rapamycintreatment (6 hours) of rat and human SMC had no effect on migration,suggesting that longer exposure to rapamycin is essential for itsanti-migratory actions. In support of these findings, acute 6 hourtreatment with rapamycin (1-100 nM), wortmannin and LY294002 of both SMCand Swiss 3T3 cells failed to inhibit PDGF-induced chemotaxis (Higaki etal., 1996). The findings that rapamycin possesses bothanti-proliferative and anti-migratory SMC properties led to thesuggestion that rapamycin may have important applications in thetreatment of disorders such as accelerated arteriopathy that occurs intransplanted hearts and restenosis after percutaneous transluminalangioplasty and placement of coronary stents (Marx et al., 1995; Marxand Marks, 1999; Poon et al., 1996). Rapamycin significantly inhibitedneointimal proliferation in a porcine angioplasty model (Gallo et al.,1999) and reversed chronic graft vascular disease in a rodent heartallograft model (Poston et al., 1999). Recent clinical studies haveimplicated the importance of rapamycin in treating stent restenosis(Sousa et al., 2000).

[0005] In p27^(kip1) (−/−) knockout mice, relative rapamycin resistancewas demonstrated, and in rapamycin resistant myogenic cells,constitutively low levels of p27^(kip1) were observed, which were notincreased with serum withdrawal and rapamycin (Luo et al., 1996). Thesefindings suggested that the ability to block p27^(kip1) down-regulationcontributes to the growth inhibitory effects of rapamycin. Transfectionof the cyclin-dependent kinase inhibitor p21^(cip1) was shown to inhibitthe spreading and attachment of SMC to extracellular matrices andmigration in a modified Boyden chamber assay. These findings suggestedthat p21^(cip1) is probably an adhesion inhibitor, as it prevented theassembly of actin filaments and the translocation of adhesion molecules(Fukui et al., 1997).

[0006] The present application discloses that rapamycin has potentinhibitory effects on SMC migration in wild type and p27 (+/−) mice, butnot in p27 (−/−) knockout mice, indicating that the cyclin-dependentkinase inhibitor (CDKI) p27^(kip1) plays a critical role in rapamycin'santi-migratory properties and in the signaling pathway(s) that regulatesSMC migration.

SUMMARY OF THE INVENTION

[0007] This invention is directed to a method of preventing migration ofa cell by increasing intracellular cyclin-dependent kinase inhibitor p27activity.

[0008] The invention provides a method of treating a subject'scardiovascular disease, which comprises administering to the subject acompound which increases intracellular cyclin-dependent kinase inhibitorp27 activity, thereby alleviating the subject's cardiovascular disease.

[0009] The invention provides a method of inhibiting tumor metastasis ina subject, which comprises administering to the subject a compound whichincreases intracellular cyclin-dependent kinase inhibitor p27 activity,thereby inhibiting tumor metastasis.

[0010] The invention provides a method of identifying a chemicalcompound that inhibits cellular migration, which comprises contactingcells whose migration is inhibited when intracellular cyclin-dependentkinase inhibitor p27 activity is increased, or contacting an extractfrom said cells, with the chemical compound under conditions suitablefor increasing p27 activity, and detecting an increase in p27 activityin the presence of the chemical compound so as to thereby identify thechemical compound as a compound which inhibits cellular migration.

[0011] The invention provides a method of screening a plurality ofchemical compounds not known to inhibit cellular migration to identify achemical compound which inhibits cellular migration, which comprises:

[0012] (a) contacting cells whose migration is inhibited whenintracellular cyclin-dependent kinase inhibitor p27 activity isincreased, or contacting an extract from said cells, with the pluralityof chemical compounds under conditions suitable for increasing p27activity;

[0013] (b) determining if p27 activity is increased in the presence ofthe plurality of chemical compounds; and if so

[0014] (c) separately determining if p27 activity is increased in thepresence of each compound included in the plurality of chemicalcompounds, so as to thereby identify any compound included therein as acompound which inhibits cellular migration.

[0015] The invention provides a chemical compound identified by any ofthe methods described herein.

[0016] The invention provides a pharmaceutical composition comprising(a) an amount of a chemical compound identified using any of the methodsdescribed herein, or a novel structural and functional homolog or analogthereof, capable of passing through a cell membrane and effective toincrease intracellular cyclin-dependent kinase inhibitor p27 activityand (b) a pharmaceutically acceptable carrier capable of passing throughthe cell membrane.

[0017] The invention provides a pharmaceutical composition comprising anamount of a chemical compound identified using any of the methodsdescribed herein effective to inhibit cellular migration and apharmaceutically acceptable carrier.

[0018] The invention provides a method for preparing a composition whichcomprises admixing a carrier and a pharmaceutically effective amount ofa chemical compound identified by any of the methods described herein ora novel structural and functional analog or homolog thereof.

[0019] The invention provides a method for making a composition ofmatter which inhibits cellular migration which comprises identifying achemical compound using any of the methods described herein, and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof.

[0020] The invention provides a method of treating a subject with acardiovascular disease which comprises administering to the subject atherapeutically effective amount of a chemical compound identified byany of the methods described herein, or a novel structural andfunctional analog or homolog thereof.

[0021] The invention provides a method of inhibiting tumor metastasis ina subject which comprises administering to the subject a therapeuticallyeffective amount of a chemical compound identified by any of the methodsdescribed herein, or a novel structural and functional analog or homologthereof.

[0022] The invention provides a use of a chemical compound identified byany of the methods described herein for the preparation of apharmaceutical composition for treating an abnormality, wherein theabnormality is alleviated by inhibiting cellular migration.

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIGS. 1A-D

[0024] Rapamycin potently inhibits migration in smooth muscle cells fromwild type, but not p27 (−/−) knockout mice.

[0025] (A) Migration of SMCs isolated from wild type mice was determinedin the modified Boyden chamber following rapamycin and FK506 treatment.Rapamycin (open bars; 1, 10 and 100 nM) significantly inhibited SMCmigration, whereas FK506 demonstrated no effect (blackened bars).*p<0.05 as compared to control. The inset shows an immunoblotdemonstrating increased p27^(kip1) levels after rapamycin (100 nM for 48hours) treatment (lane 2) as compared to untreated proliferating SMC(lane 1).

[0026] (B) Migration of SMCs isolated from p27 (−/−) knockout mice wasdetermined in the modified Boyden chamber following rapamycin and FK506treatment. Only at high concentrations did rapamycin (open bars; 100 and1000 nM) significantly inhibit SMC migration, whereas FK506 demonstratedno effect (blackened bars). *p<0.05 as compared to control. The insetshows an immunoblot demonstrating the absence of p27^(kip1).

[0027] (C and D) FK506 competes with rapamycin for binding to FKBP12 andinhibits the effects of rapamycin on wild type (C) and p27 (−/−) (D) SMCmigration.

[0028] FIGS. 2A-B

[0029] Lack of effect of rapamycin on murine SMC adhesion.

[0030] Wild type (open bars) and p27 (−/−) (blackened bars) SMC wereincubated with rapamycin for 48 hours before plating onto eitherfibronectin (A) or laminin (B) coated plates for 3 hours. The number ofadhering cells was determined with a Coulter counter in triplicate andnormalized to the number of untreated wild type cells. No significantdifferences were noted between treated and untreated cells.

[0031] FIGS. 3A-C

[0032] In vivo administration of rapamycin potently inhibits explantmigration of SMC from wild type but not p27 (−/−) knockout animals.

[0033] (A) p27 (+/+), p27 (+/−) and p27 (−/−) mice were injected withrapamycin (4 mg/kg/day) for 5 days. The aortas were explanted, andmigration of SMC was quantified and is presented as therapamycin-mediated inhibition of migration as a % of control. Rapamycinsignificantly inhibited migration in both p27 (+/+) and p27 (+/−) SMC;rapamycin had no effect on p27 (−/−) SMC explant migration

[0034] (B) p27 (+/+), p27 (+/−) and p27 (−/−) mice were injected withrapamycin (9 mg/kg/day) for 7 days. Rapamycin inhibited migration in p27(+/+), p27 (+/−) and p27 (−/−) SMC explants.

[0035] (C) p27 (+/+) and p27 (−/−) mice were injected with taxol (20mg/kg/day) for 7 days. Taxol inhibited migration in p27 (+/+) and p27(−/−) SMC.

[0036]FIG. 4

[0037] Impaired migration-inhibitory response to C3 exoenzyme in SMCderived from p27 (−/−) knockout mice.

[0038] Migration of SMC isolated from wild type mice (open bars) and p27(−/−) mice (blackened bars) was determined in the modified Boydenchamber following C3 exoenzyme (2 and 20 μg/ml) treatment for 16 hours.SMC derived from p27 (−/−) mice demonstrated a 25% relative migratoryresistance to C3 exoenzyme. *p<0.05 as compared to control.

[0039]FIG. 5

[0040] Rapamycin and C3 exoenzyme inhibit SMC migration throughp27^(kip1)-dependent and -independent pathways.

[0041] Rapamycin (Rapa)-FKBP12 inhibits target-of-rapamycin(TOR)-mediated activation/phosphorylation of protein translationmodulators 4E-BP1 (a translation initiation factor) and p70 S6 kinase(S6 is a ribosomal protein) (Marx and Marks, 1999) and preventsmitogen-induced down-regulation of p27^(kip1) through an unknownmechanism (dashed lines). Rapamycin inhibits SMC migration throughp27^(kip1)-dependent and -independent mechanisms. C3 exoenzyme, whichspecifically ADP ribosylates and inhibits RhoA, inhibits SMC migrationthrough p27^(kip1)-dependent and-independent (cytoskeleton changes)pathways.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention is directed to a method of preventingmigration of a cell by increasing intracellular cyclin-dependent kinaseinhibitor p27 activity. In different embodiments of the method, the cellis a smooth muscle cell or a tumor cell.

[0043] The invention provides a method of treating a subject'scardiovascular disease, which comprises administering to the subject acompound which increases intracellular cyclin-dependent kinase inhibitorp27 activity, thereby alleviating the subject's cardiovascular disease.In different embodiments, the cardiovascular disease is atherosclerosis,arteriopathy after heart transplantation, or restenosis afterangioplasty or coronary stent placement.

[0044] The invention provides a method of inhibiting tumor metastasis ina subject, which comprises administering to the subject a compound whichincreases intracellular cyclin-dependent kinase inhibitor p27 activity,thereby inhibiting tumor metastasis.

[0045] In one embodiment of the methods described herein,cyclin-dependent kinase inhibitor p27 activity is increased byincreasing C3 exoenzyme activity.

[0046] In different embodiments cyclin-dependent kinase inhibitor p27activity is increased by pharmacological techniques, by recombinanttechniques, or by gene therapy. Pharmacological techniques, recombinanttechniques, and gene therapy techniques are well known in the art.

[0047] The invention provides a method of identifying a chemicalcompound that inhibits cellular migration, which comprises contactingcells whose migration is inhibited when intracellular cyclin-dependentkinase inhibitor p27 activity is increased, or contacting an extractfrom said cells, with the chemical compound under conditions suitablefor increasing p27 activity, and detecting an increase in p27 activityin the presence of the chemical compound so as to thereby identify thechemical compound as a compound which inhibits cellular migration. Inone embodiment, the chemical compound is not previously known to inhibitcellular migration.

[0048] The invention provides a method of screening a plurality ofchemical compounds not known to inhibit cellular migration to identify achemical compound which inhibits cellular migration, which comprises:

[0049] (a) contacting cells whose migration is inhibited whenintracellular cyclin-dependent kinase inhibitor p27 activity isincreased, or contacting an extract from said cells, with the pluralityof chemical compounds under conditions suitable for increasing p27activity;

[0050] (b) determining if p27 activity is increased in the presence ofthe plurality of chemical compounds; and if so

[0051] (c) separately determining if p27 activity is increased in thepresence of each compound included in the plurality of chemicalcompounds, so as to thereby identify any compound included therein as acompound which inhibits cellular migration.

[0052] In different embodiments of the methods described herein,cyclin-dependent kinase inhibitor p27 activity is detected usingimmunoblots.

[0053] In different embodiments of the methods described herein, thecells are smooth muscle cells or tumor cells. In one embodiment, thecells are vertebrate cells. In a further embodiment, the vertebratecells are mammalian cells. In a still further embodiment, the mammaliancells are human cells.

[0054] The invention provides a chemical compound identified by any ofthe methods described herein.

[0055] The invention provides a pharmaceutical composition comprising(a) an amount of a chemical compound identified using any of the methodsdescribed herein, or a novel structural and functional homolog or analogthereof, capable of passing through a cell membrane and effective toincrease intracellular cyclin-dependent kinase inhibitor p27 activityand (b) a pharmaceutically acceptable carrier capable of passing throughthe cell membrane.

[0056] The invention provides a pharmaceutical composition comprising anamount of a chemical compound identified using any of the methodsdescribed herein effective to inhibit cellular migration and apharmaceutically acceptable carrier.

[0057] The invention provides a method for preparing a composition whichcomprises admixing a carrier and a pharmaceutically effective amount ofa chemical compound identified by any of the methods described herein ora novel structural and functional analog or homolog thereof.

[0058] The invention provides a method for making a composition ofmatter which inhibits cellular migration which comprises identifying achemical compound using any of the methods described herein, and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof.

[0059] The invention provides a method of treating a subject with acardiovascular disease which comprises administering to the subject atherapeutically effective amount of a chemical compound identified byany of the methods described herein, or a novel structural andfunctional analog or homolog thereof. In different embodiments, thecardiovascular disease is atherosclerosis, arteriopathy after hearttransplantation, or restenosis after angioplasty or coronary stentplacement.

[0060] The invention provides a method of inhibiting tumor metastasis ina subject which comprises administering to the subject a therapeuticallyeffective amount of a chemical compound identified by any of the methodsdescribed herein, or a novel structural and functional analog or homologthereof.

[0061] The invention provides a use of a chemical compound identified byany of the methods described herein for the preparation of apharmaceutical composition for treating an abnormality, wherein theabnormality is alleviated by inhibiting cellular migration. In differentembodiments, the abnormality is a cardiovascular disease or a tumormetastasis. In different embodiments, the cardiovascular disease isatherosclerosis, arteriopathy after heart transplantation, or restenosisafter angioplasty or coronary stent placement.

[0062] In the subject invention, a “pharmaceutically effective amount”is any amount of a compound which, when administered to a subjectsuffering from a disease against which the compound is effective, causesreduction, remission, or regression of the disease. Furthermore, as usedherein, the phrase “pharmaceutically acceptable carrier” means any ofthe standard pharmaceutically acceptable carriers. Examples include, butare not limited to, phosphate buffered saline, physiological saline,water, and emulsions, such as oil/water emulsions.

[0063] A “structural and functional analog” of a chemical compound has astructure similar to that of the compound but differing from it inrespect to a certain component or components. A “structural andfunctional homolog” of a chemical compound is one of a series ofcompounds each of which is formed from the one before it by the additionof a constant element. The term “analog” is broader than and encompassesthe term “homolog”.

[0064] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details Materials And Methods

[0065] Reagents: Dulbecco Modified Eagle Medium (DMEM) and trypsin wereobtained from GIBCO (Grand Island, N.Y.), recombinant bFGF was obtainedfrom Biosource International (Camarillo, Calif.), and paclitaxel wasobtained from Sigma (St. Louis, Mo.). Rapamycin was a gift from Dr.Suren Sehgal (Wyeth-Ayerst Laboratories, Princeton, N.J.)

[0066] Expression of C3 exoenzyme: C3 exoenzyme was prepared aspreviously described (Dillon and Feig, 1995). The Glutathione STransferase (GST)-C3 exoenzyme cDNA (gift of Dr. Judy Meinkoth,University of Pennsylvania) was transformed into competent BL21. Proteinexpression was induced with 200 μM isopropylthiogalactoside (IPTG) at32° C. for 3 hours. Lysates were prepared and incubated withGST-sepharose beads for 1 hour at 4° C. The beads were washed andincubated overnight at 4° C. with 3 units/ml thrombin (for cleavage ofthe C3 exoenzyme from the GST fusion protein), which was removed byincubating the supernatant with antithrombin-sepharose beads for 1 hourat 4° C. The supernatant was concentrated with a Centricon-10 (AmiconInc, Beverly, Mass.). Protein concentration was determined by Bradfordassay and the supernatant was aliquoted and frozen in liquid nitrogen.The samples were run on SDS-PAGE and stained with Coomassie to conformcorrect expression of the GST fusion protein and cleavage/purificationof C3 exoenzyme before use (Seasholtz et al., 1999).

[0067] Cell Culture: The murine aortic SMCs were obtained from theexplant migration experiments described below, and were subcultured inDMEM containing 20% fetal bovine serum (FBS) at 37° C. in a humidified95% air-5% CO₂ atmosphere (Kobayashi et al., 1993). The growth mediumwas changed every other day until 80% confluence was reached. The cellsused for experiments were from passages #3-6. Verification of SMCphenotype was determined by positive fluorescent staining for α-actinand negative staining for Factor VIII antigen. Cell viability was 95% orgreater as determined by trypan blue exclusion at the conclusion of eachexperiment.

[0068] SMC Adhesion Assay: The adhesion assay was performed aspreviously described (Wang et al., 1997). Murine SMCs were treated withrapamycin or vehicle for 48 hours. SMCs (5×10⁵/ml in DMEM supplementedwith 0.2% bovine serum albumin (BSA)) were loaded onto 12-well platespre-coated with laminin or fibronectin. After 3 hours, the mediacontaining nonadherent cells were removed, and cell numbers weredetermined by triplicate counts using a Coulter Counter (Model Z1,Coulter Electronics, Beds, England).

[0069] SMC migration assay: Migration was measured using a 48 wellmodified Boyden chamber housing a polycarbonate filter with 8 μm poresas described previously (Bornfeldt et al., 1994; Poon et al., 1996).Each membrane was coated with 0.1 mg/ml of collagen in 0.2 M acetic acidfor 24 hours before each assay. For each assay, 50 ng/ml of bFGF in DMEMwas loaded in quadruplicate wells in the bottom chamber. BSA (0.2% inDMEM without bFGF) was used as a negative control. Rapamycin, FK506 orC3 exoenzyme was directly added to the growth medium for either 48 hours(rapamycin and FK506) or 16 hours (C3 exoenzyme) before the cells weretrypsinized, and counted with a hemacytometer. An equal number of cells(2×10⁵/ml) in 50 μl was loaded to the top chamber of each well. After 6hours, non-migrating cells were scraped from the upper surface of thefilter. Cells on the lower surface were fixed with methanol and stainedwith Giemsa stain (Fisher Scientific, N.Y.). The number of SMC on thelower surface of the filter was determined by counting four high power(×200) fields of constant area per well. Values are expressed as thepercentage of cells migrating in response to bFGF after subtraction ofthe negative control (DMEM+BSA). Experiments were performed at leasttwice using quadruplicate wells.

[0070] Aortic SMC explant migration: Wild type C57BL/6 mice werepurchased from Jackson Laboratory (Bar Harbor, Me.). The p27 (+/−) andp27 (−/−) knockout mice were kindly provided by Dr. Andrew Koff ofMemorial Sloan-Kettering Cancer Institute (Kiyokawa et al., 1996). Themice received one of three different treatment protocols (9 mg/kg/dayfor 7 days, 4 mg/kg/day for 5 days, or 2 mg/kg/day for 2 days) ofrapamycin via intraperitoneal (IP) injection. The control group wastreated with vehicle alone (0.2% sodium CMC, polysorbate 0.25%; St.Louis, Mo.). At the conclusion of the treatment protocol, the mice wereeuthanized with 100 mg/kg of pentobarbital, the aortas excised and theadventitia and surrounding connective tissue were removed. The aortaswere then opened by a longitudinal cut and the intima, as well as a thinportion of the subjacent media, were removed. The media were dividedinto 2 mm×2 mm pieces and placed in 6 well tissue culture plates (35 mm,22.6 mm diameter, Costar, Cambridge, Mass.) containing DMEM with 20%FBS. The culture media was changed every other day. The migration of SMCout of the explant was observed under the microscope daily followingexplant. The total number of cells explanted was determined for eachanimal's explants on a daily basis. The results in FIG. 5 are presentedas the mean percentage (±SD) of inhibition of migration (by rapamycin ortaxol) as compared to control (untreated) for at least 4 animals fromeach group. The SMC phenotype was confirmed as previously described(Spector et al., 1997).

[0071] Immunoblots: Immunoblots were prepared using procedurespreviously described in Luo et al. (1996) SMC growing in log phase ortreated with rapamycin (100 nM for 48 hours) were washed twice with icecold phosphate buffered saline (PBS) and lysates prepared using amodified RIPA buffer as previously described (Poon et al., 1996).Lysates were clarified by centrifugation for 20 minutes at 14,000 rpm at4° C. Protein concentrations were determined by Bradford assay with BSAas a standard (Bradford, 1976). Protein extracts (30 μg) weresize-fractionated on SDS-12% polyacrylamide gels and transferred tonitrocellulose. Filters were blocked with PBS-0.1% Tween 20 and 5% drymilk for 1 hour at room temperature, followed by incubation with a mousemonoclonal P27^(kip1) antibody (F8 antibody, Santa Cruz BiotechnologyInc, Santa Cruz, Calif.) for 2 hours. Filters were washed with PBS-0.1%Tween 20 and then incubated with a secondary antibody conjugated toperoxidase for 1 hour. Filters were washed with PBS-0.1% Tween 20;signals were detected using chemiluminescence detection system (ECL)followed by exposure to Kodak XAR film.

[0072] Statistics: Data are presented as the mean ± standard deviation(SD) of the independent experiments. Statistical significance wasdetermined by one way analysis of variance (ANOVA) and Fisher's PLSDtest (StatView 4.01; Brain Power, Inc., Calabasas, Calif.). A paired ttest (StatView 4.01) was used to analyze all data. A p value of <0.05was considered statistically significant.

Results

[0073] The inhibitory effects of rapamycin on the migration of SMCsisolated from wild type and p27 (−/−) knockout mice were determined. Inwild type murine SMC, rapamycin treatment for 48 hours demonstrated asignificant inhibitory effect on bFGF-induced SMC migration (FIG. 1A,open bars). The inhibition was concentration dependent between 1 nM and100 nM, with an IC₅₀ of ˜2 nM. In contrast, no significant inhibition ofmigration by rapamycin (1 nM to 10 nM) was observed in the p27 (−/−) SMC(FIG. 1B, open bars). At higher concentrations (100 nM), anapproximately 35% inhibition was observed; the IC₅₀ in p27 (−/−) cellswas ˜200 nM, representing a 100 fold increased IC₅₀ as compared to wildtype SMC. Addition of rapamycin to either the upper or lower chambersimmediately prior to incubation had no effect on SMC migration. FK506,an agent that binds to the same cytosolic receptor (FKBP12) asrapamycin, had no effect on murine SMC migration (FIGS. 1A and 1B,blackened bars). The inhibition of migration of wild type murine SMC byrapamycin (10 nM) was competitively inhibited by a 100-fold molar excessof FK506 (FIG. 1C). The rapamycin-induced inhibition of migration (100nM) in the p27 (−/−) SMC was also competitively inhibited by a 20 foldmolar excess of FK506 (FIG. 1D). These data indicate that the inhibitionof migration was mediated through rapamycin's binding to FKBP12.Treatment of wild type murine SMC with rapamycin (100 nM for 48 hours)caused a significant increase in p27^(kip1) protein levels (FIG. 1A,inset); in contrast, no p27^(kip1) was detected in p27 (−/−) SMC (FIG.1B, inset). Although rapamycin inhibits SMC proliferation, thedifferences in migration do not reflect proliferation as equal numbersof cells were loaded into the Boyden chamber. To confirm this, thenumbers of cells in the upper and lower chambers after the 6 hourincubation were equal in the untreated and treated wild type and p27(−/−) SMC. In addition, no differences in cell viability were notedbetween untreated and rapamycin treated SMC obtained from wild type andp27 (−/−) animals. No morphologic differences were observed betweenuntreated and rapamycin (100 nM for 48 hours) treated SMC isolated fromwild type mice and p27 (−/−) mice.

[0074] Since migration is dependent upon the adhesion of the SMC to theBoyden chamber membrane, adhesion assays were performed usingfibronectin and laminin-coated plates. SMC obtained from p27 (−/−)animals demonstrated no differences in adhesion as compared to SMCobtained from wild type animals on both fibronectin and laminin coatedplates. Furthermore, rapamycin treatment (100 nM for 48 hours) did notaffect cell adhesion in either wild type or p27 (−/−) SMC (FIG. 2).

[0075] To assess the in vivo effects of rapamycin on SMC migration inthe p27 (−/−) animals, the ability of SMC to migrate out of the murineaortic explants and establish cell cultures was examined. Rapamycin wasnot added to the culture medium after the aortas were explanted. Explantmigration of aortic SMC was performed using wild type C57BL/6, p27(+/−), or p27 (−/−) mice. SMC from wild type, p27 (+/−) and p27 (−/−)migrated out of the aortic explant by day #2. In animals treated withrapamycin (4 mg/kg/day for 5 days), ˜85% inhibition of migration ascompared to untreated animals was observed in the wild type and p27(+/−) groups (p<0.05). In contrast, no rapamycin-mediated inhibition ofmigration was observed in p27 (−/−) group (p<0.05, FIG. 3A), indicatingthat p27^(kip1) plays a critical role in the rapamycin-mediatedinhibition of SMC migration. At higher doses (9 mg/kg/day for 7 days),equivalent levels of rapamycin-mediated inhibition of migration wereobserved in wild type, p27 (+/−) and p27 (−/−) cells (FIG. 3B). At lowerdoses (2 mg/kg/day for 2 days), no rapamycin-mediated inhibition ofmigration was observed. These results are consistent with the findingsobtained in the modified Boyden chamber for p27 (−/−) cells and suggeststhe presence of both p27^(kip1)-dependent and p27^(kip1)-independentpathways mediating rapamycin's SMC anti-migratory actions. In order todemonstrate that agents that did not perturb the p27^(kip1) pathwaycould inhibit migration in p27 (−/−) animals, wild type and p27 (−/−)animals were treated with taxol (20 mg/kg/day for 7 days) (Sollott etal., 1995). No differences in taxol-mediated inhibition were observed inthe two groups (FIG. 3C).

[0076] Recent data suggests that the Ras/RhoA mitogenic pathwayregulates the destruction of p27^(kip1). C3 exoenzyme, which adenosinediphosphate (ADP)-ribosylates and inactivates RhoA, inhibitedPDGF-induced p27^(kip1) degradation. These findings suggest thatactivation of RhoA by mitogens is necessary for degradation ofp27^(kip1) (Weber et al., 1997). In addition, thrombin-induced vascularSMC DNA synthesis and migration were inhibited by C3 exoenzyme(Seasholtz et al., 1999). We sought to determine whether this inhibitionof migration was mediated, in part, by regulating p27^(kip1) levels. SMCfrom wild type and p27 (−/−) animals were exposed to either 2 μg/ml or20 μg/ml C3 exoenzyme for 16 hours, trypsinized and loaded into theupper chamber of the Boyden chamber. C3 exoenzyme significantlyinhibited bFGF-mediated SMC migration in wild type cells (FIG. 4, openbars). SMC from p27 (−/−) animals demonstrated a 25% relative resistanceto C3 exoenzyme (FIG. 4, blackened bars). SMC that were acutely exposedto C3 exoenzyme demonstrated no inhibition of migration. These resultsimplicate p27^(kip1) as a regulator, in part, of both rapamycin and C3exoenzyme-mediated inhibition of SMC migration.

Discussion

[0077] Rapamycin has been shown previously to inhibit rat, porcine, andhuman SMC migration (Poon et al., 1996). In addition, rapamycin reducesintimal thickening by 50% after coronary angioplasty in the porcinemodel (Gallo et al., 1999). The rapamycin anti-restenotic effect ischaracterized by an inhibition of the SMC response to coronary injurywith a concomitant decrease in retinoblastoma protein (pRb)phosphorylation as well as an increase in p27^(kip1) levels, therebyresulting in cell-cycle arrest (Gallo et al., 1999; Marx et al., 1995).The cyclin-dependent kinase inhibitor (CDKI) p27^(kip1) inhibits theregulatory activities of cyclin/CDK complexes including cyclinE/CDK2 bydirectly binding to them and, in turn, blocking the phosphorylation ofretinoblastoma protein (pRb) (Kato et al., 1994; Nourse et al., 1994).Thus, p27^(Kip1) is a regulator of cell proliferation; reduction ofp27^(kip1) protein levels during the late G₁ phase is required forcyclin/CDK complex activation and cell cycle progression in certain celllines. The CDKI p27^(kip1) is present at high levels in quiescent cellsand upon mitogenic stimulation is downregulated (Kato et al., 1994;Nourse et al., 1994). Down-regulation of p27^(kip1) by mitogens can beblocked by the immunosuppressant rapamycin (Nourse et al., 1994).

[0078] The function of p27^(Kip1) is clinically relevant because of theconnections that have been made between the down-regulation and enhanceddegradation of p27^(Kip1) in colorectal, stomach, breast, and small-celllung cancers (Steeg and Abrams, 1997). Furthermore, the regulation ofthe CDKI p27^(kip1) plays a critical role in the regulation of SMCproliferation in vivo. Decreased levels of p27^(kip1) in the vessel wallhas been associated with increased neointimal response afterpercutaneous transluminal angioplasty (PTCA) (Braun-Dullaeus and al.,1997; Tanner et al., 1998). Angiotensin II stimulation of quiescentvascular SMC in which P27^(kip1) levels are high results in SMChypertrophy but induces SMC hyperplasia when levels of p27^(kip1) arelow as occurs in the presence of mitogens (Braun-Dullaeus et al., 1999).The findings disclosed in the present application suggest that agentsthat increase p27^(kip1) levels in vivo may have both ananti-proliferative and anti-migratory effect.

[0079] Although the regulation of p27^(kip1) can occur at the mRNA level(Hengst and Reed, 1996), most studies have supported the concept thatp27^(kip1) is regulated post-transcriptionally and involves ubiquin(Ub)-proteasome dependent degradation (Pagano et al., 1995). Targetingof p27^(kip1) for ubiquitin is believed to involve phosphorylation ofp27^(kip1) by cyclin E-cdk2 complex (Sheaff et al., 1997; Vlach et al.,1997). Recently, a ubiquin-proteasome independent pathway has beendescribed that involves proteolytic processing that rapidly clips offthe cyclin-binding domain. This ubiquitin independent processing isATP-dependent and sensitive to proteasome-specific and chymotrypsininhibitors (Shirane et al., 1999).

[0080] In addition, p27^(kip1) levels have been shown to be regulated bythe Ras/RhoA mitogenic pathway. Overexpression of a dominant negativeRas or RhoA inhibited the platelet derived growth factor (PDGF) induceddegradation of p27^(kip1) C3 exoenzyme, which ADP-ribosylates andinactivates RhoA, inhibited PDGF-induced p27^(kip1) degradation (Hiraiet al., 1997; Weber et al., 1997) and inhibited thrombin-mediatedvascular SMC proliferation and migration (Seasholtz et al., 1999). InSwiss 3T3 fibroblasts, it has been shown that Rho can be activated byextracellular ligands (lysophosphatidic acid) and that Rho activationcan lead to the assembly of contractile actin-myosin filaments and focaladhesion complexes (Hall, 1998). Rac, a member of the Rho subfamily, hasbeen shown to induce actin-rich surface protrusions (filopodia); Rac canactivate Rho (although in fibroblasts this is interaction is weak anddelayed) (Hall, 1998). Generation ofphosphatidylinositol-3,4,5-trisphosphate (PIP3) by PI 3-kinase activityis essential for receptor-mediated activation by Rac in mammalian cellsand a PI3 kinase homolog, TOR2 (target of rapamycin 2) controls Rholpactivation in Saccharomyces cerevisiae (Hall, 1998; Schmidt et al.,1997). These observations suggests that the Rho GTPase family is one ofthe key regulatory molecules that link surface receptors to theorganization of the actin cytoskeleton. Rapamycin has not been shown tointeract with the Rho GTPase family, although it is interesting thatinhibition of both Rho (Hirai et al., 1997; Weber et al., 1997) and mTOR(Brown et al., 1994; Nourse et al., 1994; Sabatini et al., 1994) areboth associated with increased levels of the CDKI, p27^(kip1).

[0081] The extracellular matrix (ECM) plays an essential role in theregulation of cell proliferation. Human capillary endothelial cells thatwere prevented from spreading (either mechanically or pharmacologicallywith cytochalasin or actomyosin) exhibited normal activation ofmitogen-activated kinases, but failed to progress through G1 phase(Huang et al., 1998). This shape dependent block in the cell cycle wascorrelated with a failure to down-regulate p27^(kip1), up-regulatecyclin D1 and phosphorylate pRb (Huang et al., 1998). Therefore, theaccumulation of p27^(kip1) in cells prevented from spreading suggeststhat p27^(kip1) could play a role in the shape-dependent cell cyclearrest produced by cell rounding. Signaling pathway components thatcould be responsible for transducing the accumulation of p27^(kip1)include Rho, which is involved in integrin-mediated changes in thecytoskeleton tension and shape, and the integrin-linked kinase, whichhas been shown to reduce the inhibitory actions of p27^(kip1) and topromote anchorage-independent growth (Chrzanowska-Wodnicka and Burridge,1996; Hotchin and Hall, 1995; Huang et al., 1998; Radeva et al., 1997).

[0082] The p21 CDKI (Cip1) has been shown to inhibit SMC migration invitro (Fukui et al., 1997; Witzenbichler et al., 1999). The spreadingand attachment of the p21^(Cip1) transfected rabbit aortic SMC toextracellular matrices (ECM) were inhibited compared to that of controlvector-transfected cells. Cip1 transfected SMC maintained a roundconformation on fibronectin. Moreover, p21^(Cip1) transfected SMCdemonstrated significantly reduced PDGF-BB mediated migration in amodified Boyden chamber (with fibronectin coated membranes). Therefore,p21^(cip1) probably acts as an adhesion inhibitor, since it prevents theassembly of actin filaments and the translocation of adhesion molecules(Fukui et al., 1997). Interestingly, our study indicates that inductionof p27^(kip1) with rapamycin did not affect adhesion to collagen ofeither wild type or p27 (−/−) cells.

[0083] The homeobox transcription factor Gax is expressed in quiescentvascular SMC and is down-regulated during SMC proliferation and vascularinjury (Witzenbichler et al., 1999). Gax up-regulates p21^(cip1) andinhibits vascular SMC proliferation and migration (Witzenbichler et al.,1999). p21^(cip1) mediates the growth inhibitory actions of Gax;overexpression of Gax does not have anti-proliferative or anti-migratoryeffects in cells derived from p21 (−/−) mice (Smith et al., 1997;Witzenbichler et al., 1999). Gax was unable to inhibit the migration offibroblasts which lacked p21^(cip1) (Witzenbichler et al., 1999).Transfection of a Gax cDNA inhibited PDGF-, bFGF-, and hepatocyte growthfactor-induced vascular SMC migration (Witzenbichler et al., 1999). Cellcycle arrest by either p16 or p21 is essential for Gax-inducedinhibition of migration. Interestingly, overexpression of Gax cDNA,which increases p21^(cip1), had no effect on the adhesion of cells tocollagen and vitronectin coated plates. Therefore, in contrast to thefibronection adhesion defect shown in cells transfected with p21^(cip1),cells transfected with Gax cDNA demonstrated no collagen/vitronectinadhesion defect. However, the studies reported conflicting informationregarding the effects of overexpression of p21^(cip1) on SMC migration;p21^(cip1) transfection of rabbit vascular SMC inhibited migration in afibronectin coated Boyden chamber (Fukui et al., 1997), whereasp21^(cip1) transfection in rat vascular SMC had no effect in acollagen/vitronectin Boyden chamber (Witzenbichler et al., 1999).

[0084] In conclusion, rapamysin and C3 exoenzyme inhibit smooth musclecell migration through p27^(kip1)-dependent and independent pathways(FIG. 5). This intriguing finding implicates p27^(kip1) in the signalingpathway(s) that regulate both SMC proliferation and migration.Technologies (e.g., pharmacologic, recombinant and/or gene therapy)aimed at increasing p27^(kip1) are expected to have dramatic effects onthe amelioration of restenosis after angioplasty or stent placement, oron accelerated arteriopathy after cardiac transplantation, as well as incancer therapy where cellular migration is a key element in tumormetastasis.

References

[0085] Bornfeldt, K. E., Raines, E. W., Nakano, T., Graves, L. M.,Krebs, E. G., and Ross, R. (1994). Insulin-like growth factor-I andplatelet-derived growth factor-BB induce directed migration of humanarterial smooth muscle cells via signaling pathways that are distinctfrom those of proliferation. J Clin Invest 93, 1266-1274.

[0086] Bradford, M. M. (1976). A rapid and sensitive method for thequantitation of microgram quantitites of protein utilising the principleof protein-dye binding. Anal Biochem 72, 248-254.

[0087] Braun-Dullaeus, R. C., and al., e. (1997). Loss of p27kip1 andinduction of Cdk1 in the rat carotid artery following balloon catheterinjury. In vivo and in vitro influence of rapamycin. FASEB J 11, A153(abstract).

[0088] Braun-Dullaeus, R. C., Mann, M. J., Ziegler, A., von der Leyen,H. E., and Dzau, V. J. (1999). A novel role for the cyclin-dependentkinase inhibitor p27kip1 in angiotension II-stimulated vascular smoothmuscle cell hypertrophy. J. Clin. Invest. 104, 815-823.

[0089] Brown, E., Albers, T., Shin, T., Ichikawa, K., Keith, C., Lane,W., and Schreiber, S. (1994). A mammalian protein targeted byG1-arresting rapamycin complex. Nature (London) 369, 756-758.

[0090] Cao, W., Mohacsi, P., Shorthouse, R., Pratt, R., and Morris, R.(1995). Effects of rapamycin on growth factor-stimulated vascular smoothmuscle cell DNA synthesis: inhibition of basic fibroblast growth factorand platelet-derived growth factor action and antagonism of rapamycin byFK506. Transplantation 59, 390-395.

[0091] Chrzanowska-Wodnicka, M., and Burridge, K. (1996). Rho-stimulatedcontractility drives the formation of stress fibers and focal adhesions.J. Cell Biol. 133, 1403-1415.

[0092] Clowes, A., and Schwartz, S. M. (1985). Significance of quiescentsmooth muscle migration in the injured rat carotid artery. Circ Res. 56,139-45.

[0093] Dillon, S. T., and Feig, L. A. (1995). Purification and assay ofrecombinant C3 transferase. Methods in Enzymology 256, 174-184.

[0094] Ferns, G. A. A., Raines, E. W., Sprugel, H., Motani, A. S.,Reidy, M. A., and Ross, R. (1991). Inhibition of neointimal smoothmuscle accumulation after angioplasty by an antibody to PDGF. Science253, 1129-1132.

[0095] Fukui, R., Shibata, N., Kohbayashi, E., Amakawa, M., Furutama,D., Hoshiga, M., Negoro, N., Nakakouji, T., Ii, M., Ishihara, T., andOhsawa, N. (1997). Inhibition of smooth muscle cell migration by the p21cyclin-dependent kinase inhibitor (Cip1). Atherosclerosis 132, 53-59.

[0096] Gallo, R., Padurean, A., Jayaraman, T., Marx, S. O., Roque, M.,Adelman, S., Chesebro, J., Fallon, J., Fuster, V., Marks, A. R., andBadimon, J. J. (1999). Inhibition of intimal thickening after balloonangioplasty in porcine coronary arteries by targeting regulators of thecell cycle. Circulation 99, 2164-2170.

[0097] Gregory, C., Huie, P., Billingham, M., and Morris, R. (1993).Rapamycin inhibits arterial intimal thickening caused by both alloimmuneand mechanical injury. Transplantation 55, 1409-1418.

[0098] Grotendorst, G. R., Seppa, H. E. J., Kleinman, H. K., and Martin,G. R. (1981). Attachment of smooth muscle cells to collagen and theirmigration toward platelet derived growth factor. Proc Natl Acad Sci USA78, 3669-3672.

[0099] Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science279, 509-514.

[0100] Hengst, L., and Reed, S. I. (1996). Translational control ofp27^(kip1) accumulation during the cell cycle. Science 271, 1861-1864.

[0101] Higaki, M., Sakaue, H., Ogawa, W., Kasuga, M., and Shimokado, K.(1996). Phosphatidylinositol 3-kinase-independent signal transductionpathway for platelet-derived growth factor-induced chemotaxis. J. Biol.Chem. 271, 29342-29346.

[0102] Hirai, A., Nakamura, S., Noguchi, Y., Yasuda, T., Kitagawa, M.,Tatsuno, I., Oeda, T., Tahara, K., Terano, T., Narumiya, S., Kohn, L.D., and Saito, Y. (1997). Geranylgeranylated rho small GTPase(s) areessential for the degradation of p27kip1 and facilitate the progressionfrom G1 to S phase in growth-stimulated rat FRTL-5 cells. J. Biol. Chem.272, 13-16.

[0103] Hotchin, N. A., and Hall, A. (1995). The assembly of integrinadhesion complexes requires both extracellular matrix and intracellularrho/rac GTPases. J. Cell. Biol. 131, 1857-65.

[0104] Huang, S., Chen, C. S., and Ingber, D. E. (1998). Control ofcyclin D1, p27kip1 and cell cycle progression in human capillaryendothelial cells by cell shape and cytoskeletal tension. Mol. Biol.Cell 9, 3179-3193.

[0105] Ihnatowycz, I. O., Winocour, P. D., and Moore, S. (1981). Aplatelet-derived factor chemotatic for rabbit smooth muscle cells inculture. Artery 9, 316-317.

[0106] Jawien, A., Bowen-Pope, D. F., Lindner, V., Schwartz, S. M., andClowes, A. W. (1992). Platelet-derived growth factor promotes smoothmuscle migration and intimal thickening in a rat model of balloonangioplasty. J Clin Invest. 89, 507-511.

[0107] Kato, J. M., Matsuoka, M., Polyak, K., Massague, J., and Sherr,C. J. (1994). Cyclic AMP-induced G1 phase arrest mediated by aninhibitor (p27kip1) of cyclin-dependent kinase-4 activation. Cell 79,487-496.

[0108] Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V.C., Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A.,and Koff, A. (1996). Enhanced growth of mice lacking thecyclin-dependent kinase inhibitor function of p27^(kip1). Cell 85,721-732.

[0109] Kobayashi, S., Mimura, Y., Naitoh, T., Kimura, I., and Kimura, M.(1993). Chemical structure-activity of cnidium rhizone-derivedphthalides for the competence inhibition of proliferation in primaryculture of mouse aorta smooth muscle cells. Japan J. Pharmacol 63,353-359.

[0110] Luo, Y., Marx, S. O., Kiyokawa, H., Koff, A., Massague, J., andMarks, A. R. (1996). Rapamycin resistance tied to defective regulationof p27^(kip1). Mol. Cell. Biol. 16, 6744-6751.

[0111] Marx, S. O., Jayaraman, T., Go, L. O., and Marks, A. R. (1995).Rapamycin-FKBP inhibits cell cycle regulators of proliferation invascular smooth muscle cells. Circ Res 76, 412-417.

[0112] Marx, S. O., and Marks, A. R. (1999). Cell cycle progression andproliferation despite 4BP-1 dephosphorylation. Mol Cell Biol. 19,6041-6047.

[0113] Nourse, J., Firpo, E., Flanagan, W. M., Coats, S., Polyak, K.,Lee, M., Massague, J., Crabtree, G., and Roberts, J. M. (1994).Interleukin-2-mediated elimination of the p27kip1 cyclin-dependentkinase inhibitor prevented by rapamycin. Nature (London) 372, 570-573.

[0114] Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., G., D.S., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995). Role ofthe ubiquitin-proteosome pathway in regulating abundance of thecyclin-dependent kinase inhibitor p27. Science 269, 682-685.

[0115] Pickering, J. G., Uniyal, S., Ford, C. M., Chau, T., Laurin, M.A., Chow, L. H., Ellis, C. G., Fish, J., and Chan, B. (1997). Fibroblastgrowth factor-2 potentiates vascular smooth muscle cell migration toplatelet-derived growth factor: upregulation of alpha2beta1 integrin anddisassembly of actin filaments. Circ Res. 80, 627-37.

[0116] Poon, M., Marx, S. O., Gallo, R., Badimon, J. J., Taubman, M. B.,and Marks, A. R. (1996). Rapamycin inhibits vascular smooth muscle cellmigration. J. Clin. Invest. 98, 2277-2283.

[0117] Poston, R. S., Billingham, M., Hoyt, E. G., Pollard, J.,Shorthouse, R., Morris, R. E., and Robbins, R. C. (1999). Rapamycinreverses chronic graft vascular disease in a novel cardiac allograftmodel. Circulation 100, 67-74.

[0118] Radeva, G., Petrocelli, T., Behrend, E., Leung-Hagesteijn, C.,Filmus, J., Slingerland, J., and Dedhar, S. (1997). Overexpression ofthe integrin-linked kinase promotes anchorage-independent cell cycleprogression. J. Biol. Chem. 272, 13937-13944.

[0119] Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., andSnyder, S. H. (1994). RAFT1: A mammalian protein that binds to FKBP12 ina rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78,35-43.

[0120] Schmidt, A., Bickle, M., Beck, T., and Hall, M. N. (1997). Theyeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2via the exchange factor ROM2. Cell 88, 531-542.

[0121] Schwartz, S. M. (1997). Smooth muscle migration inatherosclerosis and restenosis. J. Clin. Invest. 100, S87-98.

[0122] Seasholtz, T. M., Majumdar, M., Kaplan, D. D., and Brown, J. H.(1999). Rho and Rho kinase mediate thrombin-stimulated vascular smoothmuscle cell DNA synthesis and migration. Circ Res 84, 1186-1193.

[0123] Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M., andClurman, B. E. (1997). Cyclin E-CDK2 is a regulator of p27^(kip1). GenesDev. 11, 1464-1478.

[0124] Shirane, M., Harumiya, Y., Ishida, N., Hirai, A., Miyamoto, C.,Hatakeyama, S., Nakayama, K., and Kitagawa, M. (1999). Down-regulationof p27^(kip1) by two mechanisms, ubiquitin-mediated degradation andproteolytic processing. J Biol Chem 274, 13886-13893.

[0125] Smith, R. C., Branellec, D., Gorski, D. H., Guo, K., Perlman, H.,Dedieu, J. F., Pastore, C., Mahfoudi, A., Denefle, P., Isner, J. M., andWalsh, K. (1997). p21cip1-mediated inhibition of cell proliferation byoverexpression of the gax homeodomain gene. Genes Dev. 11, 1674-89.

[0126] Sollott, S. J., Cheng, L., Pauly, R. R., Jenkins, G. M.,Monticone, R. E., Kuzuya, M., Froehlich, J. P., Crow, M. T., Laketta, E.G., Rowinsky, E. K., and Kinsella, J. L. (1995). Taxol inhibitsneointimal smooth muscle cell accumulation after angioplasty in the rat.J. Clin. Invest. 95, 1869-1876.

[0127] Sousa, J. E., Costa, M. A., Abizaid, A., Abizaid, A. S., Feres,F., Pinto, I. M. F. et al. (2000) Lack of neointimal proliferation afterimplantation of sirolimus-coated stents in human coronary arteries. Aquantitative coronary angiography and three-dimensional intravascularultrasound study. Circulation 102, r54-r57.

[0128] Spector, D. L., Goldman, R. D., and Leinwand, L. A. (1997).Cells: a laboratory manual. (New York: Cold Spring Harbor LaboratoryPress).

[0129] Steeg, P. S., and Abrams, J. S. (1997). Cancer prognostics: Past,present and p27. Nature Med. 3, 152-154.

[0130] Tanner, F. C., Yang, Z. Y., Duckers, E., Gordon, D., Nabel, G.J., and Nabel, E. G. (1998). Expression of cyclin-dependent kinaseinhibitors in vascular disease. Circ Res 82, 396-403.

[0131] Vlach, J., Hennecke, S., and Amati, B. (1997).Phosphorylation-dependent degradation of the cyclin-dependent kinaseinhibitor p27^(kip1). EMBO J. 16, 5334-5344.

[0132] Wang, W., Chen, H. J., Warshofsky, M., Schwartz, A., C. A., S.,and L. E., R. (1997). Effects of S-dC28 on vascular smooth muscle celladhesion and plasminogen activator production. Antisense & Nucleic AcidDrug Development 7, 101-107.

[0133] Weber, J. D., Hu, W., Jefcoat, S. C., Raben, D. M., andBaldassare, J. J. (1997). Ras-stimulated extracellular signal-relatedkinase 1 and RhoA activities coordinate platelet-derived growthfactor-induced G1 progression through the independent regulation ofcyclin D1 and p27^(kip1). J Biol Chem 272, 32966-32971.

[0134] Witzenbichler, B., Kureishi, Y., Luo, Z., Le Roux, A., Branellec,D., and Walsh, K. (1999). Regulation of smooth muscle cell migration andintegrin expression by the Gax transcription factor. J. Clin. Invest.104, 1469-1480.

What is claimed is:
 1. A method of preventing migration of a cell byincreasing intracellular cyclin-dependent kinase inhibitor p27 activity.2. The method of claim 1, wherein the cell is a smooth muscle cell or atumor cell.
 3. A method of treating a subject's cardiovascular disease,which comprises administering to the subject a compound which increasesintracellular cyclin-dependent kinase inhibitor p27 activity, therebyalleviating the subject's cardiovascular disease.
 4. The method of claim3, wherein the cardiovascular disease is atherosclerosis, arteriopathyafter heart transplantation, or restenosis after angioplasty or coronarystent placement.
 5. A method of inhibiting tumor metastasis in asubject, which comprises administering to the subject a compound whichincreases intracellular cyclin-dependent kinase inhibitor p27 activity,thereby inhibiting tumor metastasis.
 6. The method of claim 1, 3, or 5,wherein cyclin-dependent kinase inhibitor p27 activity is increased byincreasing C3 exoenzyme activity.
 7. A method of identifying a chemicalcompound that inhibits cellular migration, which comprises contactingcells whose migration is inhibited when intracellular cyclin-dependentkinase inhibitor p27 activity is increased, or contacting an extractfrom said cells, with the chemical compound under conditions suitablefor increasing p27 activity, and detecting an increase in p27 activityin the presence of the chemical compound so as to thereby identify thechemical compound as a compound which inhibits cellular migration. 8.The method of claim 7, wherein the chemical compound is not previouslyknown to inhibit cellular migration.
 9. A method of screening aplurality of chemical compounds not known to inhibit cellular migrationto identify a chemical compound which inhibits cellular migration, whichcomprises: (a) contacting cells whose migration is inhibited whenintracellular cyclin-dependent kinase inhibitor p27 activity isincreased, or contacting an extract from said cells, with the pluralityof chemical compounds under conditions suitable for increasing p27activity; (b) determining if p27 activity is increased in the presenceof the plurality of chemical compounds; and if so (c) separatelydetermining if p27 activity is increased in the presence of eachcompound included in the plurality of chemical compounds, so as tothereby identify any compound included therein as a compound whichinhibits cellular migration.
 10. The method of claim 7 or 9, wherein thecells are smooth muscle cells or tumor cells.
 11. The method of claim 7or 9, wherein the cells are vertebrate cells.
 12. The method of claim11, wherein the vertebrate cells are mammalian cells.
 13. The method ofclaim 12, wherein the mammalian cells are human cells.
 14. A chemicalcompound identified by the method of claim 7 or
 9. 15. A pharmaceuticalcomposition comprising (a) an amount of a chemical compound identifiedusing the method of claim 7 or 9, or a novel structural and functionalhomolog or analog thereof, capable of passing through a cell membraneand effective to increase intracellular cyclin-dependent kinaseinhibitor p27 activity and (b) a pharmaceutically acceptable carriercapable of passing through the cell membrane.
 16. A pharmaceuticalcomposition comprising an amount of a chemical compound identified usingthe method of claim 7 or 9 effective to inhibit cellular migration and apharmaceutically acceptable carrier.
 17. A method for preparing acomposition which comprises admixing a carrier and a pharmaceuticallyeffective amount of a chemical compound identified by the method ofclaim 7 or 9 or a novel structural and functional analog or homologthereof.
 18. A method for making a composition of matter which inhibitscellular migration which comprises identifying a chemical compound usingthe method of claim 7 or 9, and then synthesizing the chemical compoundor a novel structural and functional analog or homolog thereof.
 19. Amethod of treating a subject with a cardiovascular disease whichcomprises administering to the subject a therapeutically effectiveamount of a chemical compound identified by the method of claim 7 or 9,or a novel structural and functional analog or homolog thereof.
 20. Themethod of claim 19, wherein the cardiovascular disease isatherosclerosis, arteriopathy after heart transplantation, or restenosisafter angioplasty or coronary stent placement.
 21. A method ofinhibiting tumor metastasis in a subject which comprises administeringto the subject a therapeutically effective amount of a chemical compoundidentified by the method of claim 7 or 9, or a novel structural andfunctional analog or homolog thereof.
 22. Use of a chemical compoundidentified by the method of claim 7 or 9 for the preparation of apharmaceutical composition for treating an abnormality, wherein theabnormality is alleviated by inhibiting cellular migration.
 23. The useof claim 22, wherein the abnormality is a cardiovascular disease or atumor metastasis.
 24. The use of claim 23, wherein the cardiovasculardisease is atherosclerosis, arteriopathy after heart transplantation, orrestenosis after angioplasty or coronary stent placement.